R21Phase Our preliminary data indicate that freely circulating methylated DNA can be found in the plasma of patients with esophageal cancer. Specifically, these data show that methylated alleles of the APC, HPP1 and p16 genes can be detected in the bloodstream of esophageal cancer patients. Furthermore, our preliminary data show decreased survival in patients with high plasma levels of methylated APC DNA, which also parallel relapse of malignancy. Thus, these circulating methylated nucleic acids show promise as biomarkers for the prognostication and monitoring of esophageal cancer patients. The overall scope of this proposed project will be to move these circulating biomarkers from the laboratory into the clinic. In the initial R21 phase, precise assays of gene-specific methylated plasma DNA will be developed and refined using quantitative real-time methylation-specific PCR, with the established methylation targets p16 and APC serving as templates. In addition, during this exploratory R21 phase, additional novel methylation targets will be identified in neoplastic esophageal tissues and plasma. In the second, R33 phase, novel methylation targets identified in the R21 phase will be clinically validated on a larger, independent cohort by performing clinical correlations with tissue and plasma methylation levels. Aim 1. To develop and validate accurate, robust, standardizable, and scalable quantitative real-time plasma DNA methylation assays using the established tissue and plasma methylation targets p16 and APC. Assays will be analytically validated with known standards of plasma from normal subjects spiked with known levels of genomic DNA, as well as known but blinded positive and negative patient blood samples. Aim 2. To identify and measure 20 novel methylation events in 50 primary esophageal carcinomas and 50 normal esophageal tissues. A panel of candidate genes with known cancer and outcome relevance and reported frequent methylation in gastrointestinal and other human tumors will be evaluated using real-time quantitative methylation specific PCR (MSP). Genes methylated in at least 20% of tumors, but in less than 5% of normal specimens, will be further pursued in Aim 3. Aim 3. To study the same pilot group of 50 patients for plasma methylation of target genes identified in tissues during Aim 2. Genes methylated in the plasma of greater than 10% of these patients will constitute plasma targets for clinical validation during the R33 phase of the current proposal.